Mass sensitive chemical sensing with a molecular recognition interface typically involves localisation or attachment of a first molecular species near or onto a sensor surface, which converts a subsequent localisation or binding interaction with a second molecular species into a readable signal. A mass sensitive chemical sensor can be defined as any device that allows for measurement of a property that scales proportionally to mass associated with or bound to a sensitive surface of that device. Several such sensor techniques can be utilised, such as evanescent wave-based sensors, e.g. surface plasmon resonance (SPR, which is capable of registering mass changes by the associated change in refractive index at the surface), optical waveguides (also dependent on refractive index changes associated with mass binding events), ellipsometry and acoustic wave devices (for example quartz crystal micro balances (QCMs)). These sensor approaches are well established in the art (see, for example, Biomolecular Sensors, Gizeli and Lowe. Taylor and Francis, London; 2002) and these types of instruments can be used for studies of chemical reactions in situ and for detection of certain molecules in a sample. Attachment of the first molecular species to the sensor surface can be performed by covalent coupling, adsorption or physical entrapment (e.g. in a polymer layer or by the use of a membrane). The covalent coupling can be done directly to the otherwise unmodified sensor transducer surface, to a polymer layer on the sensor surface or with the use of a chemical/biochemical “capture system”. The best approach for a particular application depends on several factors including the nature of the sample, the sensor transducer type, the manner in which the sensor will be used and the surface chemistry.
For some applications, electrostatic repulsion between the first molecular species and the sensor surface may decrease the ability of a molecule to approach and become bound to the sensor surface. This effect can reduce the sensitivity and the number of possible immobilisation strategies. In the field of biosensing, covalent coupling of biomolecules via their superficial primary amino groups to a carboxylated sensor surface has been found to be a useful and versatile immobilisation strategy. This technology can be used for the determination of specificity, concentration, affinity constants, kinetic parameters, and monitoring of multimolecular interactions in various biomolecular systems. The immobilisation of proteins to a carboxymethyldextran-modified gold surface has been previously described (Johnsson et al. Analytical biochemistry 198, 266-277 (1991)). In the first step of the immobilisation procedure a mixture of NHS(N-hydroxy succinimide) and EDC(N-ethyl-N′-(dimethylaminopropyl) carbodiimide) is passed over a carboxymethyldextran gold sensor surface. The EDC/NHS injection activates the surface due to the transformation of a proportion of the carboxyl groups into reactive esters (N-hydroxysuccinimide esters). In general, not all of the carboxyl groups are activated, thus leaving a degree of negative charge in the sensor surface matrix. In the next step, a protein (as first molecular species) dissolved in a low-ionic strength buffer at a pH below the isoelectric point (pI) of the protein is passed over the activated surface. The protein is concentrated in the matrix by electrostatic attraction between the positively charged protein and the negatively charged carboxyl groups in the matrix. During this process the NHS-esters react with the primary amino groups of the protein. The electrostatic adsorption uptake and thus also the covalent immobilisation will decrease with the buffer ionic strength. This is due to competition between the positive proteins and other positive ions in the solution. Therefore a low-ionic strength buffer is preferably used. In the last step, the remaining NHS-esters are transformed into amides by injection of ethanolamine hydrochloride. This step also removes electrostatically (i.e. non-covalently) bound material, although a subsequent optional injection of buffer or dilute acid can be employed to enhance this process. A key parameter in the covalent immobilisation is the pH of the protein solution. The covalent binding of the proteins to the active esters is favoured by a high pH (when the primary amino groups of the protein are uncharged). On the other hand, pH must be lower than the isoelectric point of the protein to achieve the electrostatic attraction of the protein to the negative carboxyl groups in the sensor surface matrix. It has generally been found that a successful immobilisation can only be performed at a pH lower than the isoelectric point of the protein, i.e. when the protein is positively charged. At the low pH value needed for relatively acidic proteins, however, both the protein binding capacity of the matrix and the reactivity of the protein is low. This immobilisation procedure has, until now, therefore been limited to proteins with pI higher than 4.
Brett et al. (Electrochem. Comm. 5, 178-183 (2003)) have described the adsorption of DNA onto pyrolytic graphite in the preparation of an electrochemical biosensor. Application of an electric potential during adsorption led to a stronger DNA-electrode surface interaction and improved DNA films were obtained in a lower pH buffer, i.e. when the charge on the DNA can be expected to be reduced. Badia et al. (Sensors and Actuators B54, 145-165 (1999)) disclose a surface plasmon spectroscopy/atomic force microscopy study of alkanethiol deposition and desorption on gold surfaces under electric potential control. Similarly, Brusatori and van Tessel (Biosensors and Bioelectronics 18, 1269-1277 (203)) employed optical waveguide light mode spectroscopy to monitor electric potential-controlled adsorption of proteins on an indium tin oxide film. Potential dependent protein adsorption has also been monitored using SPR by Schlereth (J. Electroanal. Chem. 464, 198-207 (1991)).
Heaton et al. (PNAS USA 98, 3701-3704 (2001)) have examined the hybridisation and denaturation of DNA duplexes under an applied electrostatic field using SPR. Similar experiments are reported in U.S. Pat. No. 6,203,981. In DE10049901, adjacent and independently chargeable ‘mobilisation’ electrodes and detection electrodes are used to concentrate charged analytes for detection. The applied electric field accelerates and facilitates detection (e.g. by impedance changes). The electric field-augmented binding event concerned, however, is non-covalent (e.g. DNA hybridisation), with no consideration given to the additional problems involved in achieving covalent attachment of chemical species. Ge et al. (Biosensors and Bioelectronics 18, 53-58 (2003)) have described the effect of applied potential on the covalent immobilisation of DNA on a gold surface modified with a layer of aminoethanethiol (AET) for the purpose of constructing molecular logic circuits. Such an approach is not, however, appropriate for the preparation of a sensor surface for a mass-sensitive chemical sensor—the AET layer, with its short alkyl claims, would not provide a sufficiently ordered surface for mass-sensitive chemical sensing, and non-specific binding would consequently be high. In addition, the amino-groups on the surface will make the surface highly positive, which will further increase non-specific binding effects in many applications since most proteins have a net negative charge at physiological pH. Furthermore, the effects reported using this chemical coupling scheme are likely to be at least partially due to non-covalent attachment to the surface.
In EP0395222, an SPR study is conducted on the non-covalent capture and release of polarisable species under the influence of an alternating electric potential. U.S. Pat. No. 5,858,799 describes an electrochemical study during which an applied potential is used to oxidise and/or reduce analytes at the surface of an SPR metal film.
Thus, the prior art primarily deals with the effect of applied electric field on the adsorption or other non-covalent immobilisation of chemical species at a surface. No consideration is given to the particular difficulties which are presented when covalent immobilisation is required in an ordered and controllable manner at or near a sensor surface of a mass-sensitive chemical sensor. Those methods which are concerned with covalent immobilisation are either limited to molecular species having particular charge characteristics or do not allow the control of immobilisation and surface characteristics required for chemical sensing applications.